Instability of artificially circularized chromosomes of Streptomyces lividans

Abstract

SummaryThe chromosomes of Streptomyces species are linear molecules, containing long terminal inverted repeats and covalently bound terminal proteins. These chromosomes undergo spontaneous deletions of the terminal sequences at high frequencies and become circularized in several cases examined. Artificial circularization of the Streptomyces lividans chromosome was also achieved by targeted recombination in vivo, in which the terminal inverted repeats of the chromosome were connected by a kanamycin resistance gene (aphII ). Under kanamycin selection, the circularized chromosomes harboured tandem amplifications of a 20.2 kb sequence that included the aphII gene flanked by direct repeats and deletions nearby. On release from kanamycin selection, the aphII amplifications and the neighbouring sequences were deleted from the chromosomes, rendering all the cultures kanamycin sensitive. The chloramphenicol resistance gene, which was prone to deletion in wild-type S. lividans, became much more stable in the kanamycin-sensitive derivatives. These results indicate that the telomeres and/or certain terminal sequences may be involved in the structural instability of Streptomyces chromosomes.

Forty years of genetics with Streptomyces: from in vivo through in vitro to in silico

“…The apparent association of deletion and amplification cycles with the end regions of the linear chromosome, although probably not in an obligate way, has recently given the topic a new impetus (e.g. Lin & Chen, 1997 ;Volff & Altenbuchner, 1998).…”

Terminal proteins essential for the replication of linear plasmids and chromosomes in Streptomyces

“…While certain strains of S. lividans have linear chromosomes, others have undergone DNA rearrangements that result in chromosome circularization and the loss of telomeres (Lin and Chen 1997;Volff et al 1997;Volff and Altenbuchner 2000). Like the telomeres of S. rochei linear plasmids, the 5Ј ends of S. lividans linear chromosomal DNA are protected by covalently attached protein (Lin et al 1993).…”

“…However, during these experiments, we observed rare spc r tsr s survivors (frequency ∼ 10 −3 versus our normal gene replacement frequency of 10 ); examination of several of these survivors by Southern blot analysis confirmed disruption of the tpgL gene, as shown in Figure 4, B and C, for BKKO5. When telomeres are deleted from Streptomyces linear replicons, the internal replication origin allows their propagation as circular DNA molecules (Shiffman and Cohen 1992;Lin et al 1993;Chang and Cohen 1994;Chang et al 1996;Lin and Chen 1997;Volff et al 1997;Qin and Cohen 1998;Volff and Altenbuchner 2000). Southern blot analysis of chromosomal DNA isolated from two individual survivors (BKKO5 and BKKO6) indicated that these survivors had, in fact, undergone the telomere loss (Fig.…”

“…Among the vehicles proposed to be involved in the transfer of antibiotic-resistance genes among, and from, Streptomyces species are linear extrachromosomal replicons (Davies 1994). How linear plasmids-and also the linear chromosomes found in streptomycetes-replicate their DNA and consequently reproduce the genes they carry has in recent years become a subject of increasing interest (Shiffman and Cohen 1992;Lin et al 1993;Chang and Cohen 1994;Chen 1996;Chang et al 1996;Lin and Chen 1997;Huang et al 1998;Qin and Cohen 1998;Redenbach et al 1999;Hiratsu et al 2000;Qin and Cohen 2000;Volff and Altenbuchner 2000;Yamasaki et al 2000).…”

Three functional replication origins of the linear and artificially circularized plasmid SCP1 of Streptomyces coelicolor

“…Since the three SCP1 replication loci could independently determine propagation in both circular and linear modes in Streptomyces, we tried to use a previous strategy for circularization of Streptomyces linear chromosomes (Lin & Chen, 1997) to artificially circularize SCP1 at 621-284 635 bp, which contained the three replication loci. Plasmid pSY29 was integrated by conjugal transfer into SCP1 of S. coelicolor J841 and then three clones with a phenotype of Thio S /Apr R were obtained after screening 25 colonies, indicating occurrence of a double-crossover event.…”